Preparation of riboflavin phosphate intermediates



"Patented Sept. 9, 1952 PREPARATION OF RmoFLAvm PHOSPHATE INTERMEDIATESLeo A. Fleirser, Upper Montclair, anfi Wg.iter Farkas, Nutley, N. J.,assl'gnors to Hoflmaiin- La Roche Inc., Nutley, N. J a corporation oi.

New Jersey No Drawing. Application July '1950,

' Serial No. 174,122

9 Claims. (Cl. 260*205) This invention relates to the preparation ofriboflavin phosphate intermediates useful in the synthesis of a stable,physiologically active monophosphoric acid ester of riboflavin. Thisester, especially in the form of its salts, e. g., its sodium salt, hasthe very desirable property of being much more soluble than riboflavinitself, and is, therefore, of great utility for the A flavin may bephosphorylated by chlorophosphoric acids to give various products,namely,

mono(monochlorophosphoric acid) salts and mono(diehlorophosphoric acid)salts of riboflavin monoesters of phosphoric acid, polyphos phoricacids, chlorine-containing phosphoric acid, and chlorine-containingpoly-phosphoric acids. These phosphorylated riboflavin compounds can allbe hydrolyzedto the monophosphoric acid ester of riboflavin. The newmethod disclosed in our aforementioned application for phosphorylatingthe riboflavin consists of pretreating phosphorus oxychloride (POCh).with water to form a. chlorophosphoric acid in which riboflavin issoluble and whereby it is readily phosphorylated at room temperature.The chlorophosphoric acids are readily prepared by reacting phosphorusoxychlori-de with about one to two moles of water. The treatment of thephosphorus oxychloride with water follows the course:

POCl3+H2O HOPOCl2+HCl P0013 2H2O-+ (HO) 2POC1+2HC1 Preferably, thereaction is carried outwith an excess of phosphorylating agent and for asuflicient length of time to insure that no riboflavin remainsunphosphorylated.

We have now found that certain simpler, cheaper and more readilyavailable substances than riboflavin itself may be phosphorylated by themethod disclosed in our aforesaid copending application to give newphosphorylated com' pounds from which the monophosphoric acid ester ofriboflavin can readily be synthesized. The necessity for employingexpensive riboflavin as a raw material is thereby avoided.

Thus, suitable starting materials for our new synthesis arel-D-l-ribitylamino-3,4-dlmethylbenzene, also called ribitylxylidine, and1-D-1'- ribitylamino-fi-arylazo-3A dimethyl benzene. Wehave found thatthese compounds upon reaction with a chlorophosphoric acid followed bywith the water-pretreated F001;.

2 hydrolysis of the reaction product give monophosplior-ic acid estersof ribitylxylidine and of 1-D-1'-rlbitylamino -6 aryl-azo 3,4dimethylbenzene. These may then be converted into the monoph-osph-oricacid esters of riboflavin.

The products of the phosphorylation reaction comprise monochlorophosphoric acid) salts of phosphoric acid esters andchloroplrosphoric acid esters ofl-D-l'-ribitylamino-'3,4-*dimethylbenzene and ofl-D-l'-ribitylamino-'6-arylazo- 3,4-dimethyl-benzene. The extent of thePhosphorylation depends upon the length of time the ribitylaminocompounds are allowed to react The, phosphorylation reaction involvingribitylirylidine may be written schematically as follows, although wewish it to be understood that we do not intend any theoreticalinterpretation as a limitation on our invention. The reactions givenherein set forth syntheses with postulated structures. It will beunderstood that although the outline given herein is ba'se'd upon-whatwe consider to be the most probable reactions, we do not wish to limitour invention to any specific theory of reaction or structure ofproducts.

CH; -NHCH2(CHOH):GH:OH 2HOPOC12 l CH1 CH; NHCHz(CHOH)aCHzOPOC1OH.HOP0012310 I CH3 CH3 -NHCH2(CHOH)ZCH2OPO(OH)1 one Nncmtononncmormom:

on; N ni:

wherein ArNzX is an aryl diazonium salt such pounds, II I, may then becondensed with alloxan, as benzene diazonium su1fate, p-nitroph'enyl dialloxantin, or; mixtureslthereof 1 to give monoazonium ch1oride,"6- orpchloropheriyldiazonium: phosphoric acid esters of riboflav n; IV, therechloride, and the like. I action being schematically illustrated asfollows:

V Hv DH; 7. e HCOH HCOH H2 o e oo a o=c NH 0 NH CE? V In O I IV V o Theaforementioned new monophosphoric acid Alternatively, the' riboflavinmonophosphoric esters; of .1 1-D+1' ribitylaminoefi+a11ylazo+3,4+di=-acid esters can be prepared more directly bYTeF methylbenzenes; maybemreparewas' 1-D:1eribitylaminoe6-arylazo3,4 dimethyl benacting themonophosphoric acid esters of; the

mentioned hereinbeforeiby reaction'ofihe known zenes, II, withbarbituric acid as schematically u p fl p y d aZO compounds With a rchloroillustrated. by the following. equation.

Y cn'zoromnn omororofl), HCOH; H 0 OH HC on HO OH noon- HOOH V 0,; 'IV"phosphoricacid; as illustrated-"by the following It'is to beunde'rstoodthat allth'eabovexre-l action "equations areintended to be.illustrative schematic equation; I

- of the reactions involved and not to restrictthe INHcHucHoHhcHgonriorlocig scope of ourinvention. Theillustratedtreactions' for 'exainple,- indicate thephosphate ester groupto' bean-carbon No. -5 of the ribityl chain; The

phosphate" group may,- however} in accordancepositionsof the ribitylchain-although the"5 on, I-NH CHM CH ornsonzo P 0 C1OH-H0 P 0 c1: 0 withour invention, be onany; of the available N=NAI. I I

riboflavin-5 monophosphoric acid ester with the iK/ NHCHKCHOHWHEOPWOHcomponent of the natural--rib'oflavin-enzyme sysposition" is preferredbecause -of' the identity of tern. r CH3 N=NAI The I extent") of thephosphorylation of the I II ribitylxylidine" and1--D-l-ribitylamino-6-aryl- In t case of phosphorylating t POC13azoFBA-dimethyI-benZene makes no difference as treated with two moles ofwater, the correspond- Cases-the hydrolysis Product is the Q ingreactions can be illustrated .by-the following phosphoric ac d ester ofthe intermediatesschematic equations While the aforementionedchloroph'osphoric acid Poc13+2H2o mommy-r2110] and.phosphoricacid-esters: of the-intermediates ROH+2(H0)2POCl H 0 may beisolated for purposes of purification it [R0P0(OH),].(HO)2P0C1 RoPown isnot necessary toefiect such isolation for the Where-.ROHJ represents..-l-D-1-ribity1-3,4-dipr p n f t fi l i fl vinm ph v methylbenze'rieo1;1-D-1,' ribity1 6+a 1az -3,4- phoric acid ester; Thereactionmixture-after dimeth'yl-.benzene.v v I phosphorylation of theintermediates by" the- Themo'nophosph'pric a id est s of compounds,water=pretreatedPOC13 is simply added'to several II,may-he.reducedflasillustratedby the .follow volumes of water, andallowed to stand for ing equationvto yjeldi a s new anduseful productsSeveral: hours. During residual,

the monophosph'oric acid esters of 1-D-1-ribitylamino-G-amino-3,4-dimethyl-benzene, III.

monophosphoric: 5 acid-.1 ester, 1' r unreacted water pretreatedPOCla'is completely hydrolyzedtto phosphoric and hydrochloricacidsand'thelintermediate ester: salts undergo h'y [H] drolysislitoiorm the.monophosphoric acid esters H offthe. intermediates. 2 The lattercompounds can then readilyibeiconvertedlinto the-riboflavin V H M E 20 PO OHM The:v following.- sexamples:willserve to. illustrate-i ribitylamino -6 arylazo 33 dimethylrbene III The new monophosphoric' acidesters comzenes; and;1:13-1 =-ribitylamino'-6-amin0e3;4edi=;

methyl-benzene} and their conversion 3 into riboflavin monophosphoricacid ester. The examples are intended only to illustrate and not torestrict the scope of our invention.

t exAr/iPLEi Monophosphoric lucid ester 'of1-D-1--ribitylamino-3,4-dimethyl-benzene To 61.3 grams (0.4 mole) ofphosphorus oxychloride were added slowly, with good agitation and withcooling to about room temperature,

7.2 grams (0.4 mole) of water. The mixture was stirreduntil evolution ofhydrogen chloride gas appeared mostly finished and allowed to standovernightv (about 16 hours). Then. 5.1 grams (0.02 mole) ofl-D-l-ribitylamino-3,4 dimethylbenzene (ribitylx'ylidine) wereaddedLwith stirring. Solution occurred and HCl' gas was evolved. Thesolution was. agitated at room temperature for six hours. With coolingthere were added 500 cc. of anhydrous ether. oil separated which,hardened upon trituration with fresh ether. This substance was boiledwith 250 cc. of water in order to effect hydrolysis to thestablemonophosphoric acid ester. The aqueous solution thereby obtainedwas evaporated in vacuo toa small volume and several parts of acetonewere added. The monophosphoric acid ester of l-D-l'-ribitylamino-3,4;-dimethyl-benzene precipitated as an oily solid which hardened onstanding. It was filtered, washed with acetone and ether and dried invacuo. The substance is very hygroscopic and in contradistinction toribitylxylidine itself, is highly water-soluble at room temperature.Upon potentiometric titration with sodium hydroxide it gave aninflection point at about pH 9 when two equivalents of the alkali hadbeen added. a H

When the. monophosphoric acid ester of ribitylxylidine is tobe employedfor the production of riboflavin monophosphoric acid ester, it is notnecessary to isolate the compound in the solid form. An aqueous solutionof the compound suitable for conversion into riboflavin monophosphoricacid ester was obtained by repeating the phosphorylation procedure andpouring the phosphorylation reaction solution into several volumes ofwater' (instead of adding ether) and neutralizing the resulting solutionto about pH 3 with caustic soda.

. EXAMPLEZT Monophosphoric acid ester of l-D-lFribitylamino-3,4-dimethylfi-phenylazo-benzene' To the aqueous solution of the monophosphoric acidester of ribitylxylidine obtained as described in the final paragraph ofExample 1 was added an equivalent quantity of benzene diazonium chloridesolution obtained by diaz'otizing aniline with sodium nitrite in thepresence of hydrochloric acid. The temperature was kept between and C.Red crystals of the monophosphoric acid ester of1-D-1'-ribitylamino-3,4- dimethyl-6-phenylazo-benzene began toprecipitate in about 5 minutes. The pH was maintained at about 3 forabout 6 hours by occasional addition of sodium acetate. The red crystalswere then filtered off and washed with saturated sodium chloridesolution until free of inorganic phosphates and air dried. This mixtureof sodium chloride and monophosphoric acid ester of 1 D -1' ribitylamino-3,4 dimethyl 6 6. without'further purification. The pure monophosphoricacid ester of I-D-Y-ribitylamino- 3,4-dimethy1-6-phenylazo-benzene wasobtained by extracting the mixture with butyl alcohol,

evaporating to a small volume and precipitating with ether. The productso obtained gave upon potentiometric titration withsodium hydroxide asharp point of inflection at about 'pH 9 upon addition of two moles ofthe alkali.

EXAMPLE 3 Alternative preparation of monophosphoric acid ester of1-D-1-ribitylam1ln0-3,4-dim8thyZ-6: phenylazo-benzene To 122:6 grams(0.8 mole) of phosphorus oxychloride were added slowly with goodagitation and with cooling to about room temperature, 14.4 grams (0.8mole) of water. The mixture was stirred until evolution of hydrogenchloride gas appeared mostly finished and allowed to stand overnight.Then 7.18 grams (0.02 mole) of l D l ribitylamino 3,4 dimethyl 6-phenylazo benzene were added with stirring. Solution occurred and HClgas was evolved. The solution was agitated at room temperature for sixhours. It was then slowly poured into 300 cc. of water. The temperaturerose to ISO- 0., thereby effecting hydrolysis to the stablemonophosphoric acid ester of 1-D-1'-ribitylamino-3,4- dimethyl 6phenylazo benzene. The solution was then cooled and neutralized to pH2.5 by cautious addition of 'sodium hydroxide. The monophosphoric acidester of 1-D-l-ribity:lamino 3,4 dimethyl 6 phenylazo -benzenecrystallized. saturated sodium chloride solution until. free frominorganic phosphates and air dried. In contradistinction to the parentunphosphorylated compound, the monophosphoric acid ester is highlysoluble in aqueous sodium hydroxide solution. i

A pure barium salt was prepared by suspending the sodium chloride-LD-lribitylamino-3A- dimethyl 6 phenylazo benzene monophosphoric acid estermixture in water, bringing the pH to 9 with sodium hydroxide to effectsolution and adding barium chloride solution. The insoluble barium saltof the monophosphoric acid ester precipitated and was filtered off,washed chloride-free with water, then with alcohol and ether and dried.The substance contained 5.53

per cent phosphorus (theory for CmHiiOiNPBa):

EXAMPLE 4 Monophosphoric acidesterof 1-D-1-ribitylamino3,4-dimethyl-G-amino-benzene 3.0 grams of the barium salt of themonophosphoric acid ester of 1-D-l-ribitylamino-3;4-dimethyl 6 phenylazobenzene obtained as described in thefinal paragraph of Example -3, weresuspended in 50 cc. of water and 5 cc. of 1N sulfuric acid were added toliberate the free ester. 1.0 gram of palladium-charcoal catalyst wasadded and the mixture hydrogenated at 70 C. under a pressure of 500lbs/sq. in. for two hours. The catalyst and. barium sulfate werefiltered on The solution obtained was now practically colorlessindicating complete reduction of the 1 D 1" ribitylamino 3,4. dimethyl6- phenylazo-benzene. The solution was made alkaline with NaOH, and thewater and aniline formed were distilled off under vacuum. Upon additionof alcohol to the residue, the sodium salt of the monophosphoric acidester of l-D-1'- It was filtered off and washed with ribitylamino 24;dimethyl 6 am-ino benzene precipitated;

j EXAMPLE-15,.

Q M onophosphoric acidester of riboflavin The .suspensionrin alcohol. ofthe sodium salt ofthe. monophosphoric acid. ester of 1.D--1 --ri-;bitylamino 3,4 dimethyl 6 1; amino benzene obtained as described inExample 4, wasacidified with Concentrated HCl and heated to reflux. Twograms of alloxantin'were added and the mixture boiled .under reflux withStirring for five hours; The typical yellow color, and fluorescenceofrib'ofiavin monophosphoric acidester developed. The solution wascooled, water added, and unreactedqalloxantin removed by filtration. Theester was adsorbed on fullers earth which was then-washedthoroughly'with water. The ester Waseluted with sodium hydroxide, givinga solution of :the sodium salt of riboflavin monophosphoricacid estercontaining noinorganic phos phates. Instead of employing alloxantininthe reaction; alloxan. or a mixture. of alloxan and alloxantin maybe'uscd.

EXAMPLE 6;

Alternative preparation of riboflavin monophos phoric acid esterAISOIUJ'JOH of .01-mole in 94 cc. of water of the monosodium salt of.the monophosphoric acidester. of l-D-l-ribitylamino-3,4rdimethy1-,fi-phenylazo-benzene was prepared by adding .01 mole of'NaO'Hto anaqueous suspension of .01 of a mole of the free ester-sodium chloridemixture-obtained as described in Example 3. 30 grams of sodium chloridewere added thereto re-- sulting in precipitation of the monosodium saltof themonophosphoric acid ester of the 1-D-1'-ribitylamino-3,4-dimethy1-6-phenylazo benzene whichwas filtered off,washed with saturated NaCl solution and air-dried. The dried product wasrefluxed with 3 grams of barbituric acid in a mixture of 6200. ofdioxane and 12 cc. of glacial acetic acid for 6 hours. The reactionmixture was then cooled and filtered from unreacted materialst Thefiltrate had the typical fluorescence of riboflavin monophosphoric acidester.v The substance was isolated by addition of barium chloridesolution and filtration of the resultant precipitateof the slightlysolublebarium salt of riboflavin monophosphoric acid ester;

We claim:

1. A process of phosphorylation which comprises reacting a memberselected from the group consisting ofl-D-l'-ribitylamino-3,4-dimethylbenzene and1-D-1'-ribitylamino-6-phenylazo- SA-dimethyl-benzene with a memberselected from the group consisting of I the two acids represented by theformulae (HO)2POC1' and HOPOClz;

2.-. A process of phosphorylation. which come prises reacting a memberselectedfrom the group consisting of, 1vD-l',-ribitylamino:BA-dimethyt;,benzene and. 1-D1-l'eribitylamino-6-pheny1azo- 5 by the formula(HO)zP-OC1..

3. A process of "phosphorylation which comprises reactinga memberselected from the group consisting of"1-D-1'ribitylamino-3,4+dimethylbenzene and1-D-1-ribity1aminofiephenylazo- 3,4-dimethyl-benzene with the. acidrepresented by the formula HOPOC12. H

4. A. process or phosphorylation'which comprises reacting a memberselected from the group consisting of 1-D=1'eribitylamino-3, hdimethylbenzene and 'leD 1'-ribitylamino-d-phenylazm 3,4 -dimethyl-benzene; witha member selected fr'om'thegroup consisting of the two acids representedby thjeformulae (HOMPOClandHOPOCla, andhydrolyzing. the reaction productto producev the corresponding.5.'-monophosphoric acid ester- .5. A.process, of phosphorylation which comprises reacting a member selectedfromthe group consisting of 1 1-D-1-ribitylamino-3,4-dimethyl9- benzeneand l-D-l' ribitylamino 6-phenylazo- 3,4-dimethyl-benzene with the, acidrepresented by the formula (HO) zPOCl; and hydrolyzing thereactionproduct to produce the corresponding 5monophosphoric.acid ester.

61A process .ofiphosphorylation which comprises reacting a memberselectedfrom the group consisting of 1 -D=1f' ribitylamino-3;41dimethylbenzene and l-D 1' ribitylamino-6 phenylazo- 3,4 -dimethyl-b enzene withthe, acidrepresented by the formula HOROClz, and hydrolyzin'g thereaction product to producethe. corresponding 5.- monophosphjoric acidester.

' 7. Riboflavin phosphate intermediatesv produced'according to theprocess of claiml.

8. Riboflavin phosphate intermediates produced according tothe processof claim 2.

9. Riboflavin phosphate intermediates produced according to the processof'claim 3;

r LEOZA. FLEXSER;

' WALTER'G. FARKAS;

REFERENCES CITED- 3 Thefollowing references are of record in the file ofthis vpatent.

V UNITED STATES PATENTS Number OTHER REFEREN ES Gerrard; ChemicalAbstracts,. vol; 39,- 1945, page 2734. a

1. A PROCESS OF PHOSPHORYLATION WHICH COMPRISES REACTING A MEMBERSELECTED FROM THE GROUP CONSISTING OF1-D-1''-RIBITYLAMINO-3,4-DIMETHYLBENZENE AND1-D-1''-RIBITYLAMINO-6-PHENYLAZO3,4-DIMETHYL-BENZENE WITH A MEMBERSELECTED FROM THE GROUP CONSISTING OF THE TWO ACIDS REPRESENTED BY THEFORMULAE (HO)2PCO1 AND HOPOC12.